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Clostridium acetobutylicum is both a model organism for the understanding of sporulation in solventogenic clostridia and its relationship to solvent formation and an industrial organism for anaerobic acetone-butanol-ethanol (ABE) fermentation. How solvent production is coupled to endospore formation—both stationary-phase events—remains incompletely understood at the molecular level. Specifically, it is unclear how sporulation-specific sigma factors affect solvent formation. Here the sigF gene in C. acetobutylicum was successfully disrupted and silenced. Not only σ F but also the sigma factors σ E and σ G were not detected in the sigF mutant (FKO1), and differentiation was stopped prior to asymmetric division. Since plasmid expression of the spoIIA operon ( spoIIAA - spoIIAB - sigF ) failed to complement FKO1, the operon was integrated into the FKO1 chromosome to generate strain FKO1-C. In FKO1-C, σ F expression was restored along with sporulation and σ E and σ G protein expression. Quantitative reverse transcription-PCR (RT-PCR) analysis of a select set of genes ( csfB , gpr , spoIIP , sigG , lonB , and spoIIR ) that could be controlled by σ F , based on the Bacillus subtilis model, indicated that sigG may be under the control of σ F , but spoIIR , an important activator of σ E in B. subtilis , is not, and neither are the rest of the genes investigated. FKO1 produced solvents at a level similar to that of the parent strain, but solvent levels were dependent on the physiological state of the inoculum. Finally, the complementation strain FKO1-C is the first reported instance of purposeful integration of multiple functional genes into a clostridial chromosome—here, the C. acetobutylicum chromosome—with the aim of altering cell metabolism and differentiation.

Clostridium is a diverse genus of obligate anaerobic, endospore-forming, Gram-positive bacteria. Pathogenic species such as C. botulinum, C. difficile, C. perfringens, and C. tetani produce upwards of 18% of all known bacterial toxins, thus making Clostridium the “most toxic” prokaryotic genus (37) and a significant concern to human and animal health. Cellulolytic and solventogenic species, such as C. thermocellum, C. saccharobutylicum, C. cellulolyticum, and C. acetobutylicum are some of the best-studied biomass-degrading bacteria and exhibit significant potential for renewable biofuel and chemical production (22, 25, 26, 33). Additionally, nontoxigenic, proteolytic species, such as C. sporogenes and C. novyi, are being engineered into promising chemotherapeutic vehicles, in what is called clostridial-directed prodrug therapy (31). Despite the increased interest and activity in clostridia research, key fundamental questions regarding differentiation and physiology remain which are not only significant to the general understanding of the bacterium but also necessary for exploring the broad spectrum of clostridia applications. Arguably one of the most important fundamental questions is the genetic orchestration of clostridial sporulation and its coupling to other stationary-phase phenomena (22, 33, 34). For example, solvent formation (solventogenesis) is the characteristic stationary-phase phenomenon in solventogenic clostridia, and it is tightly if not causally associated with sporulation except in a set of genetically uncharacterized mutants obtained by random mutagenesis or continuous culture (15, 16, 23, 27). Recent comparative genomics approaches and DNA microarray analyses (17, 34) have reinforced the prevailing assumption that clostridial sporulation is similar if not identical to Bacillus subtilis sporulation, but experimentally this has recently been challenged (12). In B. subtilis, sporulation is initiated by a multicomponent phosphorelay that activates Spo0A in the predivisional cell (3), which then promotes the expression of mother cell-specific and prespore-specific sigma factors σE and σF (45). Active σE regulates the expression of numerous mother cell-specific genes (9) and, with the combined activity of SpoIIID, activates the mother cell-specific σK. The combined activities and intercompartmental communication of σE and σF lead to activation of the prespore-specific σG (45), which then regulates the expression of many prespore-specific genes (51). Disruption of sigF in B. subtilis blocked sporulation at stage II, resulting in normal-looking sporulation septum but also an accumulation of disporic cells (14). The sigE disruption also blocked sporulation at stage II, resulting in a similar, disporic morphology as the sigF mutant. Disruption of sigG blocked sporulation at stage III, exhibiting engulfment but no spore cortex or coat (7). σF activation is prevented until septation (41), when SpoIIE dephosphorylates the anti-anti-sigma factor SpoIIAA, which then binds the anti-sigma factor SpoIIAB to release active σF (41). σE activity is stalled until SpoIIGA processes the pro-σE, which requires septation and a physical interaction between SpoIIGA and the σF-regulated SpoIIR (20, 24, 36). Regulation of σG appears to be complex and multilayered (4, 28). Transcriptional analysis of pH-controlled C. acetobutylicum batch cultures suggested that the orchestration of Spo0A and the major sporulation sigma factors (σF, σE, and σG) are similar to those of B. subtilis (17). Sporulation factor activities were deduced by the expression patterns of putative regulon genes. The data suggested that Spo0A activity spikes during the transition from acidogenesis to solventogenesis (∼12 h after inoculation) and remains active throughout the duration of the culture. σF and σE both exhibited a major spike in activity during mid-stationary phase (24 h after inoculation), while σG activity spiked 6 h later, at 30 h after inoculation. Regarding the control of solventogenesis, relatively little is known beyond Spo0A activity (11, 38). Spo0A induces the expression of key solventogenic genes in C. acetobutylicum (11, 47), namely, the sol locus genes organized in two operons (aad-ctfA-ctfB and adc) located on the pSOL1 megaplasmid (6, 32). Furthermore, clostridia exhibit the unique clostridial cell form (16), which is an important sporulation-associated morphology. The clostridial cell form is characteristic of all clostridia, is commonly assumed to be the solvent-producing cell type in solventogenic clostridia (16, 49), and is characterized by the accumulation of granules of a glycogen-like (1,4-glucosyl glucan) biopolymer (39). Here we examined the roles of σE and σG in C. acetobutylicum by inactivating their genes and assessed their impact on solvent production and morphogenesis of the clostridial cell form. The disruption strains were characterized by Western blot analysis, Southern blot analysis, semiquantitative reverse transcription-PCR (RT-PCR), sporulation assays, phase-contrast microscopy, electron microscopy, flow cytometry (FC), and metabolite analysis. We found significant differences between these asporogenous clostridia disruption mutants and the corresponding B. subtilis mutants. Lastly, we examined the necessity of σE and σG for granulose formation and the development of the clostridial cell form, as well as for solvent formation.

The study of microbial heterogeneity at the single-cell level is a rapidly growing area of research in microbiology and biotechnology due to its significance in pathogenesis, environmental biology, and industrial biotechnologies. However, the tools available for efficiently and precisely probing such heterogeneity are limited for most bacteria. Here we describe the development and application of flow-cytometric (FC) and fluorescence-assisted cell-sorting techniques for the study of endospore-forming bacteria. We show that by combining FC light scattering (LS) with nucleic acid staining, we can discriminate, quantify, and enrich all sporulation-associated morphologies exhibited by the endospore-forming anaerobe Clostridium acetobutylicum . Using FC LS analysis, we quantitatively show that clostridial cultures commonly perform multiple rounds of sporulation and that sporulation is induced earlier by the overexpression of Spo0A, the master regulator of endospore formers. To further demonstrate the power of our approach, we employed FC LS analysis to generate compelling evidence to challenge the long-accepted view in the field that the clostridial cell form is the solvent-forming phenotype.

Mass yields of biofuels and chemicals from sugar fermentations are limited by the decarboxylation reactions involved in Embden-Meyerhof-Parnas (EMP) glycolysis. This paper reviews one route to recapture evolved CO2 using the Wood-Ljungdahl carbon fixation pathway (WLP) in a process called anaerobic, non-photosynthetic (ANP) mixotrophic fermentation. In ANP mixotrophic fermentation, the two molecules of CO2 and eight electrons produced from glycolysis are used by the WLP to generate three molecules of acetyl-CoA from glucose, rather than the two molecules that are produced by typical fermentation processes. In this review, we define the bounds of ANP mixotrophy, calculate the potential metabolic advantages, and discuss the viability in a number of host organisms. Additionally, we highlight recent accomplishments in the field, including the recent discovery of electron bifurcation in acetogens, and close with recommendations to realize mixotrophic biofuel and biochemical production.

The primary alcohol/aldehyde dehydrogenase (coded by the aad gene) is responsible for butanol formation in Clostridium acetobutylicum. We complemented the non-sporulating, non-solvent-producing C. acetobutylicum M5 strain (which has lost the pSOL1 megaplasmid containing aad and the acetone-formation genes) with aad expressed from the phosphotransbutyrylase promoter and restored butanol production to wild type levels. Because no acetone was produced, no acids (acetate or butyrate) were re-assimilated leading to high butyrate but especially acetate levels. To counter acetate production, we examined thiolase overexpression in order reduce the acetyl-CoA pool and enhance the butyryl-CoA pool. We combined thiolase overexpression with aad overexpression aiming to also enhance butanol formation. While limiting the formation of acetate and ethanol, the butanol titers were not improved. We also generated acetate kinase (AK) and butyrate kinase (BK) knockout (KO) mutants of M5 using a modified protocol to increase the antibiotic-resistance gene expression. These strains exhibited greater than 60% reduction in acetate or butyrate formation, respectively. We complemented the AKKO M5 strain with aad overexpression, but could not successfully transform the BKKO M5 strain. The AKKO M5 strain overexpressing aad produced less acetate, but also less butanol compared to the M5 aad overexpression strain. These data suggest that loss of the pSOL1 megaplasmid renders cells resistant to changes in the two acid-formation pathways, and especially so for butyrate formation. We argue that the difficulty in generating high butanol producers without acetone and acid production is hindered by the inability to control the electron flow, which appears to be affected by unknown pSOL1 genes.